Ken Muneoka

Cellular contribution from dermis and cartilage to the regenerating limb blastema in axolotls.

Using the triploid/diploid cell marker in the axolotl, Ambystoma mexicanum, we have analyzed the extent to which cells derived from the dermis and the skeleton contribute to the regenerating limb blastema. We found that dermal cells contribute 43% of the blastemal cell population whereas cells derived from skeletal tissue contribute only 2%. When compared to the availability of cells at the plane of amputation, dermal cells overcontribute by greater than twofold whereas skeletal cells undercontribute by several-fold. These data correlate with the effects that these two tissues have on the formation of the limb pattern during regeneration; dermis has a dramatic influence on pattern and skeletal tissue has virtually no effect. It is suggested that the fibroblasts present in the dermis and in other parts of the limb form virtually all of the mesodermal tissues in the regenerate with the exception of the muscle.

The migration of dermal cells during blastema formation in axolotls

Using the diploid/triploid cell marker in the axolotl (Ambystoma mexicanum) we have examined the movement of cells from the dermis into the early limb blastema. Cells of dermal origin begin to migrate beneath the wound epithelium at about 5 days postamputation, and by 10 days they are widely distributed across the amputation surface. By 15 days, a dense accumulation of blastema cells is present beneath the apical cap, and these cells are preferentially oriented in a circumferential direction. These results are discussed in relation to previous studies showing that the progeny of dermal cells become widely distributed during regeneration, and that cells of dermal origin are a major source of blastema cells. The results are also discussed in relation to ideas about how growth and patterning of the new appendage occur.

Cellular contribution to supernumerary limbs resulting from the interaction between developing and regenerating tissues in the axolotl

The relationship between limb development and limb regeneration is considered with regard to the mechanisms by which pattern is established during limb outgrowth. In a previous paper (Muneoka, K. and Bryant, S. V. 1982 Nature (London) 298, 369-371) the interaction between cells from the developing limb bud and the regenerating limb blastema was found to result in the production of organized supernumerary limb structures. In this paper the relative cellular contribution from developing and regenerating cells to supernumerary limbs resulting from contralateral grafts between limb buds and blastemas has been analyzed using the triploid cell marker in the axolotl. Results show that there is substantial participation from both developing and regenerating limb cells to all supernumerary limbs analyzed. These data lend further support to the hypothesis that developing and regenerating limbs utilize the same patterning mechanisms during limb outgrowth. This conclusion is discussed in terms of patterning models for developing and regenerating limbs and it is proposed that the rules of the polar coordinate model can best explain the behavior of cells during limb development as well as limb regeneration.

Views of limb development and regeneration

Abstract Features of vertebrate limb development and limb regeneration are interpreted from two different perspective: the zone of polarizing activity model and the polar coordinate model.

Cellular contribution to supernumerary limbs in the axolotl, Ambystoma mexicanum

Using the triploid cell marker, the cellular contribution from graft and stump to the supernumerary limbs which result from controlateral grafts of limb buds and regeneration blastemas in the axolotl has been analyzed. Grafts were made so as to appose anterior and posterior limb positions. Overall, the contribution from graft and stump tissue was found to be approximately equal although the position of the boundary between the two was variable from limb to limb. This result is consistent with models which suggest that intercalary regeneration is the driving force for patterning of the vertebrate limb. In addition, the pattern of cellular contribution to supernumerary limbs was consistently found to be asymmetrical in the dorsal-ventral axis. Hence, posterior limb tissue predominantly contributed cells to the posterior and dorsal part of the supernumerary limb whereas anterior limb tissue predominantly contributed cells to the anterior and ventral part of the supernumerary limb. The reason for this asymmetrical pattern remains unknown, but we suggest that it might result from a directional bias in intercalary regeneration, similar to that observed during intercalation in the proximal-distal axis of the urodele limb. Using the triploid cell marker in conjunction with a black/white pigmentation marker, the relationship between the cellular contribution boundary and the pigmentation boundary in supernumerary limbs has also been analyzed. It has been found that the positions of the two boundaries do not coincide, a result which suggests that the eventual location of pigment cells is not a good indicator of the location of nonpigment cells derived from graft and stump.

Improved techniques for use of the triploid cell marker in the axolotl, Ambystoma mexicanum.

Techniques for using the triploid cell marker for studying cell lineage during the development and regeneration of the axolotl limb are described. Triploid animals possess cells with three nucleoli while diploid animals possess cells with two nucleoli. We have developed a technique for isolating the limb dermis as a sheet of cells for whole-mount analysis of cellular ploidy. Whole-mount tissue preparations as well as paraffin-embedded sectioned tissues were stained specifically for nucleoli with bismuth. Cell counts from a number of triploid and diploid dermal preparations show that (1) diploid dermal cells never possess three nucleoli, (2) the frequency of trinucleolate cells in whole-mount triploid dermal preparations is not 100% but varies between animals from 30 to 76%, (3) within a single triploid animal, the frequency of trinucleolate cells in different dermal preparations is constant. These data establish the usefulness of this technique and emphasize the need for appropriate control cell counts when using the triploid cell marker in the axolotl.